FIG. 4.
Dia2 protein turnover is not controlled by the SCF pathway. (A) N-terminal deletion mutants of Dia2 are stabilized. Dia2Myc strains were treated with α-factor for 2 h and translation inhibited by CHX. Cells were collected at the indicated time points after inhibition, and protein samples were resolved by SDS-PAGE prior to immunoblotting with anti-myc antibodies. Pgk1 was monitored as a loading control. Quantitation of results from three independent experiments is shown on the graph. Error bars indicate standard deviations. (B) Turnover of Dia2 does not require the SCF pathway. The 9MYC-DIA2 allele was inserted into skp1-11, cdc53-1, and cdc34-2 strains. Cultures were grown in YPD at 25°C and shifted to 37°C for 2 h prior to the addition of CHX. Samples were collected at the indicated times and immunoblotted with anti-myc or anti-Pgk1 antibodies. Quantitation of three independent experiments is shown on the graph. (C) Deletion of the N-terminal region mislocalizes Dia2. Dia2 variants were expressed in dia2Δ cells, and protein was visualized by immunofluorescence as described in Materials and Methods. Scale bar, 5 μm.